This is an amended application, which examines the molecular mechanisms that regulate the migration of leukocytes from blood vessels to inflammatory loci in airways. The activation of integrin-regulated signaling pathways involved in cell spreading, migration, and secretion of toxic granular proteins are important determinants of the severity of inflammation. Pyk2, a member of the focal adhesion kinase family, transfers signals from integrins to downstream signaling cascades that regulate cell behavior. Preliminary investigations suggest that Pyk2 has no effect on integrin-adhesion;however, Pyk2 is required for subsequent eosinophil spreading and migration. The central hypothesis of this proposal is that Pyk2 regulates eosinophil spreading and migration subsequent to integrin ligation by reorganization of cytoskeletal structures. In Aim 1, eosinophils are used as model cells to determine the potential role of Pyk2 in leukocyte migration. Studies are proposed to determine the role of Pyk2 in regulation of eosinophil spreading and migration by specific inhibition of Pyk2 using 1) dominant negative Pyk2 fused to a TAT-protein transduction domain and 2) lentivirus-mediated transfection of Pyk2 interfering RNA and various mutants of Pyk2. Specific functional domains of Pyk2 that are required for eosinophil migration will be identified in these studies. Additional studies are proposed to determine the potential mechanisms by which Pyk2 regulates actin cytoskeletal organization, with particular focus on actin-binding proteins, gelsolin and paxillin. To validate the potential role of Pyk2 in the orchestration of the acute inflammatory response in vivo, studies are proposed in Aim 2 to examine the role of Pyk2 in airway inflammation and airway hyperresponsiveness to methacholine caused by antigen challenge in a mouse model. On this amended application, studies are added to examine the effect of TAT-dominant negative Pyk2 on infiltration of eosinophils, lymphocytes and macrophages in murine airways. The effect of Pyk2 inhibition on airway hyperresponsiveness and epithelial cell hyperplasia after ovalbumin challenge will also be examined. These studies will establish the relevance of signaling pathways for Pyk2, as elucidated in Aims 1 in migration of inflammatory cells in immune-sensitized mouse model of asthma. Data derived from these studies should suggest novel approaches for anti-inflammatory therapies in asthma and atopic diseases. PUBLIC HEALTH RELEVANCE. Studies are proposed to determine the mechanisms by which Pyk2, a protein tyrosine kinase, regulates eosinophil migration in vitro and in a mouse model of asthma. Data derived from these studies should suggest novel approaches for anti-inflammation therapies in asthma and other allergic diseases.